Plant underground propagule excitant

Abstract

The invention discloses a plant underground propagule excitant, and belongs to the technical field of excitant preparation. The plant underground propagule excitant is composed of 169.91-196.13 parts of a mixture A and 2-4 parts of an immobilized microbial agent, and the mixture A is composed of the following components in parts by weight: 0.1-0.2 part of brassinolide, 1-2 parts of sodium nitroprusside, 30-40 parts of cane sugar, 70-80 parts of urea, 68-73 parts of disodium hydrogen phosphate and 0.81-0.93 part of a composite bioactive matter. According to the plant underground propagule excitant prepared by the method, the grassland improvement effect exceeds the expectation, the aboveground biomass is increased by 1-2.5 times, the root biomass is increased by 2-3 times, and the density of vegetation plants (clusters) is increased by 2-3 times.

Description

Technical Field

[0001] The invention belongs to the technical field of stimulant preparation, and in particular relates to a plant underground propagule stimulant. Background Art

[0002] Grassland resources in my country account for 40% of the country's land area. Due to over-exploitation and climate change over the years, more than 90% of available natural grasslands have degraded to varying degrees. Degraded grasslands are characterized by insufficient coverage, lack of species, and an increase in forbs. Increasing the density and potential of underground propagules has become a key plant factor in grassland restoration. Including the density and potential of underground asexual bud banks, timely germination and growth of seeds in soil seed banks have become key technologies for regulating grassland restoration capabilities. The "ecological package" technology proposed based on the concept of plant diversity in China has successfully achieved grassland restoration in some areas. Internationally, the use of native species reseeding and disturbance has promoted the research and development of theories and technologies for multi-species reseeding. However, native grass seeds have the characteristics of dormancy, slow germination, and uneven germination, which seriously affect the effect of grassland restoration. For large-scale ecological restoration practices, the low quality of seeds and the difficulty of mechanized sowing have led to a single reseeding community composition, poor stability, low seed emergence and survival rate, and difficulty in promoting reseeding technology, which have become bottlenecks restricting degraded grassland restoration technology. Therefore, developing plant underground propagule stimulants can promote the rapid germination and growth of compound reseeding seeds, stimulate the native seed bank, and form a stable and diverse community, which will greatly enhance the technology of multi-species combination reseeding to improve grassland. Summary of the invention

[0003] The purpose of the present invention is to provide a plant underground propagule stimulator. Aiming at the problems of insufficient propagules and weak vitality in degraded grasslands, based on the common physiological ecology theory of tillering and germination, through multi-dimensional regulation such as plant hormone combination, signal factor induction, and microbial agent regulation, a common promoter of underground propagules is developed to increase the density and vitality of bud banks, break seed dormancy, promote rapid germination, and provide theoretical and technical support for the rapid restoration of degraded grassland vegetation.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] The invention provides a plant underground propagule stimulator, characterized in that the plant underground propagule stimulator consists of 169.91-196.13 parts of a mixture A and 2-4 parts of an immobilized bacterial agent, wherein the mixture A consists of the following components in parts by weight: 0.1-0.2 parts of brassinolide, 1-2 parts of sodium nitroprusside, 30-40 parts of sucrose, 70-80 parts of urea, 68-73 parts of disodium hydrogen phosphate, and 0.81-0.93 parts of a composite biologically active substance, wherein the composite biologically active substance consists of dehydroabietic acid 、 Sulfonyl chloride 、 Pyridine、 Thiosemicarbazide 、 It is prepared by sodium hydroxide and benzyl chloride, and the immobilized bacterial agent is made of lipoic acid 、 Triethylenetetramine 、 N,N'-Carbonyldiimidazole 、 L-Arginine 、 The method is prepared by using N-acetyl-L-cysteine ​​methyl ester and microbial agents.

[0006] Preferably, the method for preparing the composite bioactive substance comprises the following steps:

[0007] Q1: Add dehydroabietic acid to a container containing dichloromethane, stir and mix, then dropwise add thionyl chloride, heat to reflux, and after reflux, perform vacuum rotary evaporation to obtain intermediate 1;

[0008] Q2: Pyridine, triethylamine and thiosemicarbazide are added to a container in sequence, stirred to dissolve, intermediate 1 is evenly mixed with pyridine, and then added dropwise to the container to react at room temperature. After the reaction is completed, distilled water is added, filtered, and dried to obtain intermediate 2;

[0009] Q3: Add intermediate 2 and sodium hydroxide solution to a container, heat to reflux for reaction, and after the reaction is completed, acidify, filter, and purify to obtain intermediate 3; add intermediate 3 and anhydrous ethanol to a container, mix and stir, then add sodium carbonate and benzyl chloride, heat to reflux, and after the reflux is completed, purify to obtain a composite bioactive substance.

[0010] In the above process, dehydroabietic acid is used as a raw material, and a composite bioactive substance is prepared through chlorination, nucleophilic substitution reaction, and coupled cyclization reaction. The addition of the composite bioactive substance can promote the synthesis and transportation of cytokinins or auxins, thereby promoting the growth and development of plants. By regulating hormone balance, the division and elongation of plant cells are stimulated to promote plant growth. The addition of the composite bioactive substance can improve the absorption capacity of plant roots and increase the transport efficiency of nutrient elements in plants, thereby improving the absorption and utilization of key nutrient elements such as nitrogen, phosphorus, and potassium by plants. At the same time, the addition of the composite bioactive substance can also change the metabolic pathways and rates in plants by regulating enzyme activity, thereby promoting the growth and development process of plants. The synthetic reaction formula of the composite bioactive substance is as follows:

[0011]

[0012] The results of mass spectrometry analysis of intermediate 1 were: m / z: 318.18 (100.0%), 320.17 (32.0%), 319.18 (22.0%), 321.18 (7.1%), 320.18 (2.5%); the results of mass spectrometry analysis of intermediate 2 were: m / z: 373.22 (100.0%), 374.22 (24.7%), 375.21 (4.5%), 375.23 (2.5%), 376.22 (1.1%); the results of mass spectrometry analysis of intermediate 3 ... The analysis results are: m / z: 355.21 (100.0%), 356.21 (25.0%), 357.20 (4.5%), 357.21 (2.9%), 358.21 (1.1%); the results of mass spectrometry analysis of the composite bioactive substances are: m / z: 445.26 (100.0%), 446.26 (30.7%), 447.26 (5.1%), 447.25 (4.5%), 446.25 (1.9%), 448.25 (1.4%).

[0013] Preferably, in Q1, the dosage ratio of dehydroabietic acid, dichloromethane and thionyl chloride is (6-8) g: (50-55) mL: (8-10.5) mL, the container contains a thermometer and a condenser reflux tube, the heating reflux temperature is 50-60°C, and the time is 2-4 h; in Q2, the molar ratio of pyridine, triethylamine, semicarbazide, intermediate 1 and pyridine mixed with intermediate 1 is (0.62-0.93): (1-1.5): (1-1.4): (1-1.5): (0.25-0.37), the stirring and dissolving time is 30-45 min, and the reaction time at room temperature is 1-2 h.

[0014] Preferably, in Q3, the dosage ratio of the intermediate 2 to the sodium hydroxide solution is (2-2.5) g: (100-120) mL, the volume fraction of the sodium hydroxide solution is 5vt%, the heating reflux temperature is 50-60°C, the time is 3-4h, and the acetic acid is used to acidify to pH = 4-5. During the purification process, the eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1; the molar ratio of the intermediate product 3, anhydrous ethanol, sodium carbonate and benzyl chloride is (2-3): (1.3-1.9): (1-1.5): (2-3), the heating reflux temperature is 60-70°C, the time is 2-3h, and the eluent during the purification process is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0015] Preferably, the method for preparing the immobilized bacterial agent comprises the following steps:

[0016] S1: Add lipoic acid and N,N'-carbonyldiimidazole to dichloromethane, stir well in an ice-water bath to obtain solution 1, add triethylenetetramine to dichloromethane, stir and dissolve to obtain solution 2, add solution 2 dropwise to solution 1, continue stirring after the addition is complete, then transfer to room temperature for reaction, wash, dry, concentrate and purify after the reaction is complete to obtain a yellow oil 1;

[0017] S2: Add lipoic acid and N,N'-carbonyldiimidazole to N,N-dimethylformamide and stir at room temperature to obtain solution 3; add L-arginine and N,N-diisopropylethylamine to N,N-dimethylformamide and stir at room temperature to obtain solution 4; add solution 4 dropwise to solution 3 and stir at room temperature to react; after the reaction is completed, add dropwise to ether and centrifuge to collect the precipitate; wash, purify and obtain a yellow oil 2;

[0018] S3: Add N-acetyl-L-cysteine ​​methyl ester to triethanolamine buffer, introduce nitrogen, then add yellow oil 1 and yellow oil 2, vigorously stir to cause polymerization reaction, and after the reaction is completed, dialyze to obtain a carrier polymer, and shake and mix the carrier polymer and the microbial agent to obtain an immobilized agent.

[0019] In the above process, lipoic acid monomer modified by triethylenetetramine and L-arginine is first synthesized, and then a carrier polymer is obtained through a ring-opening polymerization reaction, which is mixed with a microbial agent to obtain an immobilized agent. After the microorganism and the carrier are compounded, the microorganism is protected from damage by environmental factors. The carrier polymer provides a good growth environment for the microorganism, which helps to maintain its activity and metabolic function. The addition of the microbial agent can help the underground propagule resist adversity and increase its survival rate and growth rate in an unfavorable environment. At the same time, the addition of microorganisms can not only improve soil fertility, but also increase the diversity of soil microorganisms, help maintain the ecological balance of the soil, and improve the self-repairing ability of the soil. The carrier polymer is degradable and will not pollute the environment. It is green and environmentally friendly. The synthetic reaction formula of the carrier polymer is as follows:

[0020]

[0021] The results of mass spectrometry analysis of the yellow oil 1 were: m / z: 334.19 (100.0%), 335.19 (17.1%), 336.18 (9.1%), 336.19 (1.8%), 337.19 (1.5%), 335.18 (1.5%); the results of mass spectrometry analysis of the yellow oil 2 were: m / z: 362.14 (100.0%), 363.15 (15.6%), 364.14 (9.1%), 363.14 (3.1%), 364.15 (2.2%), 365.14 (1.6%).

[0022] Preferably, in S1, the molar ratio of lipoic acid, N,N'-carbonyldiimidazole and triethylenetetramine is (1-2): (1.32-2.64): (7.75-15.5), the stirring time is 15-30 min, the continued stirring time is 1-2 h, the reaction time is 1-2 h, the mixture is washed with saturated sodium chloride and dried with anhydrous sodium sulfate, and the eluent in the purification process is a mixed solution of methanol and dichloromethane in a volume ratio of 1:40.

[0023] Preferably, in S2, the molar ratio of lipoic acid, N,N'-carbonyldiimidazole, L-arginine and N,N-diisopropylethylamine is (1-2):(1-1.9):(0.5-0.9):(0.5-0.8), the stirring time at room temperature is 2-3h, the stirring reaction time at room temperature is 30-45min, the temperature of ether is 0-1°C, and washing is performed with a mixed solution of methanol and ether in a volume ratio of 1:2.

[0024] Preferably, in S3, the molar ratio of N-acetyl-L-cysteine ​​methyl ester, yellow oil 1 and yellow oil 2 is (0.03-0.05):(1-1.1):(0.9-1.2), the polymerization reaction time is 1-2h, the dialysis is carried out in ultrapure water for 7-9 days, and the mass ratio of the carrier polymer to the microbial agent is (10-15):(0.5-1).

[0025] Preferably, the method for preparing the plant underground propagule stimulator comprises the following steps:

[0026] Step 1: mixing brassinolide, sodium nitroprusside, sucrose, urea, disodium hydrogen phosphate and the composite bioactive substance to obtain a mixture A;

[0027] Step 2: Mixture A and immobilized bacterial agent together form a plant underground propagule stimulator.

[0028] Preferably, the application method of the plant underground propagule stimulator is: during the spring thawing period, the plant underground propagule stimulator is applied to the 3-4 cm soil layer. During the application process, mixture A is applied first, and the immobilized bacterial agent is added after 48 hours. The row spacing of the plant underground propagule stimulator is 10-20 cm, and the application amount is 10-20 kg / mu.

[0029] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0030] The plant underground propagule stimulator prepared by the present invention can target and change the composition and dosage of soil microorganisms and soil chemical nutrients, and coordinately regulate soil properties in multiple dimensions to achieve increased root dough, nutrient efficiency, increased vegetation diversity, and improved grassland productivity. On the one hand, the ratio of sucrose, urea and nitrification inhibitor provides energy and nitrogen source for microorganisms, and the nitrification inhibitor simultaneously regulates nitrogen hydrolysis, nitrification and denitrification in the soil, balances the content of ammonium nitrogen and nitrate nitrogen in the soil, reduces ammonia volatilization and nitrate nitrogen leaching, increases nitrogen utilization efficiency, increases effective nitrogen content in the soil, and prolongs the effectiveness time; on the other hand, sodium nitroprusside as a nitric oxide source and brassinolide in a certain proportion promote the density of plant underground bud banks, and further, under the regulation of nitrification inhibitor, the nitrogen content and phosphorus content and proportion in the soil promote the root growth of grasses, the dominant population of grassland, and the phosphate fertilizer and brassinolide synergistically promote the germination of dormant seeds in the soil; at the same time, the addition of the added composite biological active substances and immobilized bacterial agents helps to increase the stress resistance of plants, improve the soil environment, and promote plant growth. The plant underground reproductive body stimulator has exceeded expectations in improving grasslands, increasing aboveground biomass by 1-2.5 times, root biomass by 2-3 times, and vegetation plant (clump) density by 2-3 times. DETAILED DESCRIPTION

[0031] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1: This example discloses a method for preparing a composite bioactive substance, comprising the following steps:

[0033] Q1: Add 7 g of dehydroabietic acid to a container containing 52.5 mL of dichloromethane equipped with a thermometer and a condenser reflux tube, stir and mix, then dropwise add 9.25 mL of thionyl chloride, heat and reflux at 55°C for 4 h, and after the reflux is completed, evaporate under reduced pressure to obtain intermediate 1;

[0034] Q2: 1 mL of pyridine, 2.44 mL of triethylamine and 1.75 g of thiosemicarbazide were added to the container in sequence and stirred for 45 min. 6.36 g of intermediate 1 was mixed with 0.4 mL of pyridine and then added dropwise to the container. The mixture was reacted at room temperature for 2 h. After the reaction was completed, distilled water was added, the mixture was filtered and dried to obtain intermediate 2.

[0035] Q3: 2.25 g of intermediate 2 and 110 mL of 5 vt% sodium hydroxide solution were added to a container, heated to reflux at 60 ° C for 4 h, and after the reaction was completed, acidified with acetic acid to pH = 4, filtered and purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, and intermediate 3 was obtained; 4.44 g of intermediate 3 and 0.47 mL of anhydrous ethanol were added to a container, mixed and stirred, and 0.67 g of sodium carbonate and 1.47 mL of benzyl chloride were added. The mixture was heated to reflux at 70 ° C for 2 h. After the reflux was completed, it was purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1 to obtain a composite bioactive substance.

[0036] This embodiment discloses a method for preparing an immobilized bacterial agent, comprising the following steps:

[0037] S1: 0.31 g lipoic acid and 0.32 g N,N'-carbonyldiimidazole were added to 10 mL dichloromethane, and stirred for 30 min in an ice-water bath to obtain solution 1. 1.73 mL triethylenetetramine was added to 10 mL dichloromethane, and stirred to dissolve to obtain solution 2. Solution 2 was added dropwise to solution 1. After the addition was completed, stirring was continued for 1 h, and then the mixture was transferred to room temperature and reacted for 2 h. After the reaction was completed, the mixture was washed with saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and purified. The eluent in the purification process was a mixed solution of methanol and dichloromethane in a volume ratio of 1:40 to obtain a yellow oil 1.

[0038] S2: 1.5 g lipoic acid and 1.14 g N,N'-carbonyldiimidazole were added to 20 mL N,N-dimethylformamide, and stirred at room temperature for 3 h to obtain solution 3. 0.59 g L-arginine and 0.4 g N,N-diisopropylethylamine were added to 20 mL N,N-dimethylformamide, and stirred at room temperature to obtain solution 4. Solution 4 was added dropwise to solution 3, and stirred at room temperature for 45 min. After the reaction, the solution was added dropwise to 0°C ether, centrifuged, and the precipitate was collected. The precipitate was washed with a mixed solution of methanol and ether in a volume ratio of 1:2, purified, and purified to obtain a yellow oil 2;

[0039] S3: 0.035 g of N-acetyl-L-cysteine ​​methyl ester was added to 2 mL of triethanolamine buffer. After nitrogen was introduced, 1.71 g of yellow oil 1 and 1.60 g of yellow oil 2 were added and the polymerization reaction was vigorously stirred for 2 h. After the reaction was completed, the mixture was dialyzed in ultrapure water for 7 days to obtain a carrier polymer. 12.5 g of the carrier polymer was mixed with 0.75 g of a microbial agent (purchased from Qingdao Kashino Import and Export Co., Ltd.) by shaking to obtain an immobilized agent.

[0040] The present embodiment discloses a plant underground propagule stimulator, which is composed of the following ingredients in parts by weight: 0.15 parts of brassinolide, 1.5 parts of sodium nitroprusside, 35 parts of sucrose, 75 parts of urea, 70.5 parts of disodium hydrogen phosphate, 0.87 parts of composite biologically active substances, and 3 parts of immobilized bacterial agents.

[0041] This embodiment discloses a method for preparing a plant underground propagule stimulator, comprising the following steps:

[0042] Step 1: mixing brassinolide, sodium nitroprusside, sucrose, urea, disodium hydrogen phosphate and the composite bioactive substance to obtain a mixture A;

[0043] Step 2: Mixture A and immobilized bacterial agent together form a plant underground propagule stimulator.

[0044] This embodiment discloses an application method of a plant underground propagule stimulator: during the spring thawing period, the plant underground propagule stimulator is applied to a 3.5 cm soil layer. During the application process, mixture A is applied first, and then an immobilized bacterial agent is added 48 hours later. The plant underground propagule stimulator is applied at a row spacing of 15 cm and an application amount of 15 kg / mu.

[0045] Example 2: This example discloses a method for preparing a composite bioactive substance, comprising the following steps:

[0046] Q1: Add 6 g of dehydroabietic acid into a container containing 50 mL of dichloromethane equipped with a thermometer and a condenser reflux tube, stir and mix, then dropwise add 8 mL of thionyl chloride, heat and reflux at 55°C for 4 h, and after the reflux is completed, evaporate under reduced pressure to obtain intermediate 1;

[0047] Q2: 0.8 mL of pyridine, 2.2 mL of triethylamine and 1.46 g of thiosemicarbazide were added to the container in sequence and stirred for 45 min. 5.09 g of intermediate 1 was mixed with 0.32 mL of pyridine and then added dropwise to the container. The mixture was reacted at room temperature for 2 h. After the reaction was completed, distilled water was added, the mixture was filtered and dried to obtain intermediate 2.

[0048] Q3: 2g of intermediate 2 and 100mL of 5vt% sodium hydroxide solution were added to a container, heated to reflux at 60℃ for 4h, and after the reaction, acidified with acetic acid to pH=4, filtered and purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, and intermediate 3 was obtained; 3.55g of intermediate 3 and 0.38mL of anhydrous ethanol were added to a container, mixed and stirred, and 0.53g of sodium carbonate and 1.18mL of benzyl chloride were added. The mixture was heated to reflux at 70℃ for 2h. After the reflux, it was purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1, and a composite bioactive substance was obtained.

[0049] This embodiment discloses a method for preparing an immobilized bacterial agent, comprising the following steps:

[0050] S1: 0.21 g lipoic acid and 0.21 g N,N'-carbonyldiimidazole were added to 10 mL dichloromethane, and stirred for 30 min in an ice-water bath to obtain solution 1. 1.15 mL triethylenetetramine was added to 10 mL dichloromethane, and stirred to dissolve to obtain solution 2. Solution 2 was added dropwise to solution 1. After the addition was completed, stirring was continued for 1 h, and then the mixture was transferred to room temperature and reacted for 2 h. After the reaction was completed, the mixture was washed with saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and purified. The eluent in the purification process was a mixed solution of methanol and dichloromethane in a volume ratio of 1:40 to obtain a yellow oil 1.

[0051] S2: 1 g lipoic acid and 0.79 g N,N'-carbonyldiimidazole were added to 20 mL N,N-dimethylformamide, and stirred at room temperature for 3 h to obtain solution 3. 0.42 g L-arginine and 0.31 g N,N-diisopropylethylamine were added to 20 mL N,N-dimethylformamide, and stirred at room temperature to obtain solution 4. Solution 4 was added dropwise to solution 3, and stirred at room temperature for 45 min. After the reaction, the solution was added dropwise to 0°C ether, centrifuged, and the precipitate was collected. The precipitate was washed with a mixed solution of methanol and ether in a volume ratio of 1:2, purified, and purified to obtain a yellow oil 2.

[0052] S3: Add 0.027 g of N-acetyl-L-cysteine ​​methyl ester to 2 mL of triethanolamine buffer, introduce nitrogen, then add 1.63 g of yellow oil 1 and 1.37 g of yellow oil 2, and vigorously stir to allow polymerization to proceed for 2 h. After the reaction is complete, dialyze in ultrapure water for 7 days to obtain a carrier polymer. Shake and mix 12.5 g of the carrier polymer with 0.75 g of the microbial agent to obtain an immobilized agent.

[0053] The present embodiment discloses a plant underground propagule stimulator, which is composed of the following ingredients in parts by weight: 0.1 parts of brassinolide, 1 parts of sodium nitroprusside, 30 parts of sucrose, 70 parts of urea, 68 parts of disodium hydrogen phosphate, 0.81 parts of composite biologically active substances, and 2 parts of immobilized bacterial agents.

[0054] This embodiment discloses a method for preparing a plant underground propagule stimulator, comprising the following steps:

[0055] Step 1: mixing brassinolide, sodium nitroprusside, sucrose, urea, disodium hydrogen phosphate and the composite bioactive substance to obtain a mixture A;

[0056] Step 2: Mixture A and immobilized bacterial agent together form a plant underground propagule stimulator.

[0057] This embodiment discloses an application method of a plant underground propagule stimulator: during the spring thawing period, the plant underground propagule stimulator is applied to a 3.5 cm soil layer. During the application process, mixture A is applied first, and then an immobilized bacterial agent is added 48 hours later. The plant underground propagule stimulator is applied at a row spacing of 15 cm and an application amount of 15 kg / mu.

[0058] Example 3: This example discloses a method for preparing a composite bioactive substance, comprising the following steps:

[0059] Q1: Add 8 g of dehydroabietic acid into a container containing 55 mL of dichloromethane equipped with a thermometer and a condenser reflux tube, stir and mix, then dropwise add 10.5 mL of thionyl chloride, heat and reflux at 55°C for 4 h, and after the reflux is completed, evaporate under reduced pressure to obtain intermediate 1;

[0060] Q2: 1.2 mL of pyridine, 3.33 mL of triethylamine and 2.04 g of thiosemicarbazide were added to the container in sequence and stirred for 45 min. 7.63 g of intermediate 1 was mixed with 0.48 mL of pyridine and then added dropwise to the container. The mixture was reacted at room temperature for 2 h. After the reaction was completed, distilled water was added, the mixture was filtered and dried to obtain intermediate 2.

[0061] Q3: 2.5 g of intermediate 2 and 120 mL of 5 vt% sodium hydroxide solution were added to a container, heated to reflux at 60 ° C for 4 h, and after the reaction, acidified with acetic acid to pH = 4, filtered and purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, and intermediate 3 was obtained; 5.33 g of intermediate 3 and 0.56 mL of anhydrous ethanol were added to a container, mixed and stirred, and 0.8 g of sodium carbonate and 1.76 mL of benzyl chloride were added. The mixture was heated to reflux at 70 ° C for 2 h. After the reflux, it was purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1 to obtain a composite bioactive substance.

[0062] This embodiment discloses a method for preparing an immobilized bacterial agent, comprising the following steps:

[0063] S1: 0.41 g lipoic acid and 0.43 g N,N'-carbonyldiimidazole were added to 10 mL dichloromethane, and stirred for 30 min in an ice-water bath to obtain solution 1. 2.31 mL triethylenetetramine was added to 10 mL dichloromethane, and stirred to dissolve to obtain solution 2. Solution 2 was added dropwise to solution 1. After the addition was completed, stirring was continued for 1 h, and then the mixture was transferred to room temperature and reacted for 2 h. After the reaction was completed, the mixture was washed with saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and purified. The eluent in the purification process was a mixed solution of methanol and dichloromethane in a volume ratio of 1:40 to obtain a yellow oil 1.

[0064] S2: 2 g lipoic acid and 1.49 g N,N'-carbonyldiimidazole were added to 20 mL N,N-dimethylformamide, and stirred at room temperature for 3 h to obtain solution 3. 0.76 g L-arginine and 0.5 g N,N-diisopropylethylamine were added to 20 mL N,N-dimethylformamide, and stirred at room temperature to obtain solution 4. Solution 4 was added dropwise to solution 3, and stirred at room temperature for 45 min. After the reaction was completed, the solution was added dropwise to 0°C ether, centrifuged, and the precipitate was collected. The precipitate was washed with a mixed solution of methanol and ether in a volume ratio of 1:2, purified, and purified to obtain a yellow oil 2;

[0065] S3: 0.044 g of N-acetyl-L-cysteine ​​methyl ester was added to 2 mL of triethanolamine buffer. After nitrogen was introduced, 1.79 g of yellow oil 1 and 1.82 g of yellow oil 2 were added. The polymerization reaction was vigorously stirred for 2 h. After the reaction was completed, the mixture was dialyzed in ultrapure water for 7 days to obtain a carrier polymer. 15 g of the carrier polymer was shaken and mixed with 1 g of the microbial agent to obtain an immobilized agent.

[0066] The present embodiment discloses a plant underground propagule stimulator, which is composed of the following ingredients in parts by weight: 0.2 parts of brassinolide, 2 parts of sodium nitroprusside, 40 parts of sucrose, 80 parts of urea, 73 parts of disodium hydrogen phosphate, 0.93 parts of composite biologically active substances, and 4 parts of immobilized bacterial agents.

[0067] This embodiment discloses a method for preparing a plant underground propagule stimulator, comprising the following steps:

[0068] Step 1: mixing brassinolide, sodium nitroprusside, sucrose, urea, disodium hydrogen phosphate and the composite bioactive substance to obtain a mixture A;

[0069] Step 2: Mixture A and immobilized bacterial agent together form a plant underground propagule stimulator.

[0070] This embodiment discloses an application method of a plant underground propagule stimulator: during the spring thawing period, the plant underground propagule stimulator is applied to a 3.5 cm soil layer. During the application process, mixture A is applied first, and then an immobilized bacterial agent is added 48 hours later. The plant underground propagule stimulator is applied at a row spacing of 15 cm and an application amount of 15 kg / mu.

[0071] Example 4: This example discloses a method for preparing a composite bioactive substance, comprising the following steps:

[0072] Q1: Add 6.5 g of dehydroabietic acid into a container containing 51 mL of dichloromethane equipped with a thermometer and a condenser reflux tube, stir and mix, then dropwise add 9 mL of thionyl chloride, heat and reflux at 55°C for 4 h, and after the reflux is completed, evaporate under reduced pressure to obtain intermediate 1;

[0073] Q2: 0.88 mL of pyridine, 2.5 mL of triethylamine and 1.6 g of thiosemicarbazide were added to the container in sequence and stirred for 45 min. 5.68 g of intermediate 1 was mixed with 0.35 mL of pyridine and then added dropwise to the container. The mixture was reacted at room temperature for 2 h. After the reaction was completed, distilled water was added, the mixture was filtered and dried to obtain intermediate 2.

[0074] Q3: 2.1 g of intermediate 2 and 105 mL of 5 vt% sodium hydroxide solution were added to a container, heated to reflux at 60 ° C for 4 h, and after the reaction was completed, acidified with acetic acid to pH = 4, filtered and purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, and intermediate 3 was obtained; 2.91 g of intermediate 3 and 0.42 mL of anhydrous ethanol were added to a container, mixed and stirred, and 0.55 g of sodium carbonate and 1.23 mL of benzyl chloride were added. The mixture was heated to reflux at 70 ° C for 2 h. After the reflux was completed, it was purified. During the purification process, the eluent was a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:1 to obtain a composite bioactive substance.

[0075] This embodiment discloses a method for preparing an immobilized bacterial agent, comprising the following steps:

[0076] S1: 0.26 g lipoic acid and 0.28 g N,N'-carbonyldiimidazole were added to 10 mL dichloromethane, and stirred for 30 min in an ice-water bath to obtain solution 1. 1.51 mL triethylenetetramine was added to 10 mL dichloromethane, and stirred to dissolve to obtain solution 2. Solution 2 was added dropwise to solution 1. After the addition was completed, stirring was continued for 1 h, and then the mixture was transferred to room temperature and reacted for 2 h. After the reaction was completed, the mixture was washed with saturated sodium chloride, dried with anhydrous sodium sulfate, concentrated, and purified. The eluent in the purification process was a mixed solution of methanol and dichloromethane in a volume ratio of 1:40 to obtain a yellow oil 1.

[0077] S2: 1.1 g lipoic acid and 0.97 g N,N'-carbonyldiimidazole were added to 20 mL N,N-dimethylformamide, and stirred at room temperature for 3 h to obtain solution 3. 0.48 g L-arginine and 0.45 g N,N-diisopropylethylamine were added to 20 mL N,N-dimethylformamide, and stirred at room temperature to obtain solution 4. Solution 4 was added dropwise to solution 3, and stirred at room temperature for 45 min. After the reaction, the solution was added dropwise to 0°C ether, centrifuged, and the precipitate was collected. The precipitate was washed with a mixed solution of methanol and ether in a volume ratio of 1:2, purified, and purified to obtain a yellow oil 2;

[0078] S3: Add 0.031 g of N-acetyl-L-cysteine ​​methyl ester to 2 mL of triethanolamine buffer, introduce nitrogen, then add 1.65 g of yellow oil 1 and 1.42 g of yellow oil 2, vigorously stir and allow the polymerization to react for 2 h. After the reaction, dialyze in ultrapure water for 7 days to obtain a carrier polymer. 11 g of the carrier polymer and 0.6 g of the microbial agent are shaken and mixed to obtain an immobilized agent.

[0079] The present embodiment discloses a plant underground propagule stimulator, which is composed of the following ingredients in parts by weight: 0.12 parts of brassinolide, 1.2 parts of sodium nitroprusside, 32 parts of sucrose, 72 parts of urea, 69 parts of disodium hydrogen phosphate, 0.84 parts of composite biologically active substances, and 2.5 parts of immobilized bacterial agents.

[0080] This embodiment discloses a method for preparing a plant underground propagule stimulator, comprising the following steps:

[0081] Step 1: mixing brassinolide, sodium nitroprusside, sucrose, urea, disodium hydrogen phosphate and the composite bioactive substance to obtain a mixture A;

[0082] Step 2: Mixture A and immobilized bacterial agent together form a plant underground propagule stimulator.

[0083] This embodiment discloses an application method of a plant underground propagule stimulator: during the spring thawing period, the plant underground propagule stimulator is applied to a 3.5 cm soil layer. During the application process, mixture A is applied first, and then an immobilized bacterial agent is added 48 hours later. The plant underground propagule stimulator is applied at a row spacing of 15 cm and an application amount of 15 kg / mu.

[0084] Comparative Example 1: Compared with Example 1, in the process of preparing the plant underground propagule stimulator in Comparative Example 1, no composite biologically active substance is added, and other conditions remain unchanged.

[0085] Comparative Example 2: Compared with Example 1, in the process of preparing the plant underground propagule stimulator in Comparative Example 2, no immobilized bacterial agent is added, and other conditions remain unchanged.

[0086] Comparative Example 3: Compared with Example 1, in Comparative Example 3, only urea and disodium hydrogen phosphate are used in the process of preparing the plant underground propagule stimulator, and other conditions remain unchanged.

[0087] Experimental example: Experimental location: grassland of Duolun County Grass Storage Station; Grassland type: haymaking field; Operation time: March 25, 2019. The treated natural grassland was hayed in autumn according to the traditional method. After one treatment, it was continuously used for 5 years. The experimental results are shown in the following table:

[0088] Table 1: Effects of different samples on forage yield in 5 years (unit: kg / mu)

[0089] project 2019 2020 2021 2022 2023 average Example 1 273 236 221 174 168 214.4 Comparative Example 1 200 187 166 133 128 162.8 Comparative Example 2 183 172 161 121 117 150.8 Comparative Example 3 82 91 92 111 85 92.2

[0090] From the test results in Table 1, it can be seen that the plant underground propagule stimulator prepared by the present invention can effectively increase the yield of forage. From the comparison between Comparative Example 1 and Example 1, it can be seen that the addition of composite biological active substances can effectively increase the yield of forage; from the comparison between Comparative Example 2 and Examples 1-4, it can be seen that the addition of immobilized bacterial agents can effectively increase the yield of forage.

[0091] Table 2: Effects of different samples on the root biomass of grassland in 5 years (unit: kg / mu)

[0092] project 2019 2020 2021 2022 2023 average Example 1 545 481 442 453 358 455.8 Comparative Example 1 326 278 272 265 234 275 Comparative Example 2 318 316 293 246 227 280 Comparative Example 3 175 148 172 181 174 170

[0093] From the test results in Table 2, it can be seen that the plant underground propagule stimulator prepared by the present invention can effectively increase the root biomass. From the comparison between Comparative Example 1 and Example 1, it can be seen that the addition of composite biological active substances can effectively increase the root biomass; from the comparison between Comparative Example 2 and Examples 1-4, it can be seen that the addition of immobilized bacterial agents can effectively increase the root biomass.

[0094] Table 3: Effects of different samples on the density of grass plants (clumps) in 5 years (unit: plants (clumps) / square meter)

[0095] project 2019 2020 2021 2022 2023 average Example 1 267 245 232 201 194 227.8 Comparative Example 1 151 183 142 141 120 147.4 Comparative Example 2 168 163 148 140 126 149 Comparative Example 3 95 88 92 91 88 90.8

[0096] From the test results in Table 3, it can be seen that the plant underground propagule stimulator prepared by the present invention can effectively increase the density of grass plants (clumps). From the comparison between Comparative Example 1 and Example 1, it can be seen that the addition of composite biological active substances can effectively increase the density of grass plants (clumps); from the comparison between Comparative Example 2 and Examples 1-4, it can be seen that the addition of immobilized bacterial agents can effectively increase the density of grass plants (clumps).

[0097] Comprehensive analysis of various indicators showed that plant underground reproductive body stimulants greatly improved grassland forage yield, increased underground root biomass and increased bud bank density.

[0098] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0099] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A plant underground propagule stimulator, characterized in that: The plant underground propagule stimulator is composed of 169.91-196.13 parts of mixture A and 2-4 parts of immobilized bacterial agents, wherein the mixture A is composed of the following components in weight: 0.1-0.2 parts of brassinolide, 1-2 parts of sodium nitroprusside, 30-40 parts of sucrose, 70-80 parts of urea, 68-73 parts of disodium hydrogen phosphate, 0.81-0.93 parts of composite biological active substances, and the composite biological active substances are dehydroabietic acid 、 Sulfonyl chloride 、 Pyridine 、 Thiosemicarbazide 、 It is prepared by sodium hydroxide and benzyl chloride, and the immobilized bacterial agent is made of lipoic acid 、 Triethylenetetramine 、 N,N'-Carbonyldiimidazole 、 L-Arginine 、 The method is prepared by using N-acetyl-L-cysteine ​​methyl ester and microbial agents.

2. The plant underground propagule stimulator according to claim 1, characterized in that The preparation method of the composite bioactive substance comprises the following steps: Q1: Add dehydroabietic acid to a container containing dichloromethane, stir and mix, then dropwise add thionyl chloride, heat to reflux, and after reflux, perform vacuum rotary evaporation to obtain intermediate 1; Q2: Pyridine, triethylamine and thiosemicarbazide are added to a container in sequence, stirred to dissolve, intermediate 1 is evenly mixed with pyridine, and then added dropwise to the container to react at room temperature. After the reaction is completed, distilled water is added, filtered, and dried to obtain intermediate 2; Q3: Add intermediate 2 and sodium hydroxide solution to a container, heat to reflux for reaction, and after the reaction is completed, acidify, filter, and purify to obtain intermediate 3; add intermediate 3 and anhydrous ethanol to a container, mix and stir, then add sodium carbonate and benzyl chloride, heat to reflux, and after the reflux is completed, purify to obtain a composite bioactive substance.

3. The plant underground propagule stimulator according to claim 2, characterized in that: In the Q1, the dosage ratio of dehydroabietic acid, dichloromethane and thionyl chloride is (6-8) g: (50-55) mL: (8-10.5) mL, the container contains a thermometer and a condenser reflux tube, the heating reflux temperature is 50-60° C., and the time is 2-4 h. In the Q2, the molar ratio of pyridine, triethylamine, thiosemicarbazide, intermediate 1 and pyridine mixed with intermediate 1 is (0.62-0.93): (1-1.5): (1-1.4): (1-1.5): (0.25-0.37), the stirring and dissolving time is 30-45 min, and the reaction time at room temperature is 1-2 h.

4. The plant underground propagule stimulator according to claim 2, characterized in that: In the Q3, the dosage ratio of the intermediate 2 to the sodium hydroxide solution is (2-2.5) g: (100-120) mL, the volume fraction of the sodium hydroxide solution is 5vt%, the heating reflux temperature is 50-60°C, the time is 3-4h, and the pH is acidified to 4-5 with acetic acid. During the purification process, the eluent is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1; the molar ratio of the intermediate product 3, anhydrous ethanol, sodium carbonate and benzyl chloride is (2-3): (1.3-1.9): (1-1.5): (2-3), the heating reflux temperature is 60-70°C, the time is 2-3h, and the eluent during the purification process is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 10:

1.

5. The plant underground propagule stimulator according to claim 1, characterized in that: The preparation method of the immobilized bacterial agent comprises the following steps: S1: Add lipoic acid and N,N'-carbonyldiimidazole to dichloromethane, stir well in an ice-water bath to obtain solution 1, add triethylenetetramine to dichloromethane, stir and dissolve to obtain solution 2, add solution 2 dropwise to solution 1, continue stirring after the addition is complete, then transfer to room temperature for reaction, wash, dry, concentrate and purify after the reaction is complete to obtain a yellow oil 1; S2: Add lipoic acid and N,N'-carbonyldiimidazole to N,N-dimethylformamide and stir at room temperature to obtain solution 3; add L-arginine and N,N-diisopropylethylamine to N,N-dimethylformamide and stir at room temperature to obtain solution 4; add solution 4 dropwise to solution 3 and stir at room temperature to react; after the reaction is completed, add dropwise to ether and centrifuge to collect the precipitate; wash, purify and obtain a yellow oil 2; S3: Add N-acetyl-L-cysteine ​​methyl ester to triethanolamine buffer, introduce nitrogen, then add yellow oil 1 and yellow oil 2, vigorously stir to cause polymerization reaction, and after the reaction is completed, dialyze to obtain a carrier polymer, and shake and mix the carrier polymer and the microbial agent to obtain an immobilized agent.

6. The plant underground propagule stimulator according to claim 5, characterized in that: In the S1, the molar ratio of lipoic acid, N,N'-carbonyldiimidazole and triethylenetetramine is (1-2): (1.32-2.64): (7.75-15.5), the stirring time is 15-30 minutes, the stirring time is continued for 1-2 hours, the reaction time is 1-2 hours, the mixture is washed with saturated sodium chloride and dried with anhydrous sodium sulfate, and the eluent in the purification process is a mixed solution of methanol and dichloromethane in a volume ratio of 1:

40.

7. The plant underground propagule stimulator according to claim 5, characterized in that: In the S2, the molar ratio of lipoic acid, N,N'-carbonyldiimidazole, L-arginine and N,N-diisopropylethylamine is (1-2): (1-1.9): (0.5-0.9): (0.5-0.8), the stirring time at room temperature is 2-3h, the stirring reaction time at room temperature is 30-45min, the temperature of ether is 0-1°C, and washing is performed with a mixed solution of methanol and ether in a volume ratio of 1:

2.

8. The plant underground propagule stimulator according to claim 5, characterized in that: In S3, the molar ratio of N-acetyl-L-cysteine ​​methyl ester, yellow oil 1 and yellow oil 2 is (0.03-0.05): (1-1.1): (0.9-1.2), the polymerization reaction time is 1-2 hours, the dialysis is carried out in ultrapure water for 7-9 days, and the mass ratio of the carrier polymer to the microbial agent is (10-15): (0.5-1).

9. The plant underground propagule stimulator according to claims 1-8, characterized in that: The preparation method of the plant underground propagule stimulator comprises the following steps: Step 1: mixing brassinolide, sodium nitroprusside, sucrose, urea, disodium hydrogen phosphate and the composite bioactive substance to obtain a mixture A; Step 2: Mixture A and immobilized bacterial agent together form a plant underground propagule stimulator.

10. The plant underground propagule stimulator according to claims 1-9, characterized in that: The application method of the plant underground propagule stimulator is as follows: during the spring thawing period, the plant underground propagule stimulator is applied to the 3-4 cm soil layer. During the application process, the mixture A is applied first, and the immobilized bacterial agent is added 48 hours later. The row spacing of the plant underground propagule stimulator is 10-20 cm, and the application amount is 10-20 kg / mu.

Images